A number of polypeptide growth factors and hormones mediate their cellular effects through a signal transduction pathway. Transduction of signals from the cell surface receptors for these ligands to intracellular effectors frequently involves phosphorylation or dephosphorylation of specific protein substrates by regulatory protein serine/threonine kinases (PSTK) and phosphatases. Serine/threonine phosphorylation is a major mediator of signal transduction in multicellular organisms. Receptor-bound, membrane-bound and intracellular PSTKs regulate cell proliferation, cell differentiation and signalling processes in many cell types.
Aberrant protein serine/threonine kinase activity has been implicated or is suspected in a number of pathologies such as rheumatoid arthritis, psoriasis, septic shock, bone loss, many cancers and other proliferative diseases. Accordingly, serine/threonine kinases and the signal transduction pathways which they are part of are potential targets for drug design.
A subset of PSTKs are involved in regulation of cell cycling. These are the cyclin-dependent kinases or CDKs (Peter and Herskowitz, Cell 1994: 79, 181-184). CDKs are activated by binding to regulatory proteins called cyclins and control passage of the cell through specific cell cycle checkpoints. For example, CDK2 complexed with cyclin E allows cells to progress through the G1 to S phase transition. The complexes of CDKs and cyclins are subject to inhibition by low molecular weight proteins such as p16 (Serrano et al, Nature 1993: 366, 704), which binds to and inhibits CDK4. Deletions or mutations in p16 have been implicated in a variety of tumors (Kamb et al, Science 1994: 264, 436-440). Therefore, the proliferative state of cells and diseases associated with this state are dependent on the activity of CDKs and their associated regulatory molecules. In diseases such as cancer where inhibition of proliferation is desired, compounds that inhibit CDKs may be useful therapeutic agents. Conversely, activators of CDKs may be useful where enhancement of proliferation is needed, such as in the treatment of immunodeficiency.
YAK1, a PSTK with sequence homology to CDKs, was originally identified in yeast as a mediator of cell cycle arrest caused by inactivation of the cAMP-dependent protein kinase PKA (Garrett et al, Mol Cell Biol. 1991: 11, 4045-4052). YAK1 kinase activity is low in cycling yeast but increases dramatically when the cells are arrested prior to the S-G2 transition. Increased expression of YAK1 causes growth arrest in yeast cells deficient in PKA. Therefore, YAK1 can act as a cell cycle suppressor in yeast.
Our U.S. Pat. No. 6,323,318 describes two novel human homologs of yeast YAK1 termed hYAK3-2, one protein longer than the other by 20 amino acids. hYAK3-2 proteins (otherwise reported as REDK-L and REDK-S in Blood, 1 May 2000, Vol 95, No. 9, pp 2838) are primarily localized in the nucleus. hYAK-2 proteins (hereinafter simply referred as hYAK3 or hYAK3 proteins) are present in hematopoietic tissues, such as bone marrow and fetal liver, but the RNA is expressed at significant levels only in erythroid or erthropoietin (EPO)-responsive cells. Two forms of REDK cDNAs appear to be alternative splice products. Antisense REDK oligonucleotides promote erythroid colony formation by human bone marrow cells, without affecting colony-forming unit (CFU)-GM, CFU-G, or CFU-GEMM numbers. Maximal numbers of CFU-E and burst-forming unit-erythroid were increased, and CFU-E displayed increased sensitivity to suboptimal EPO concentrations. The data indicate that REDK acts as a brake to retard erythropoiesis. Thus inhibitors of hYAK3 proteins are expected to stimulate proliferation of cells in which it is expressed. More particularly, inhibitors of hYAK3 proteins are useful in treating or preventing diseases of the erythroid and hematopoietic systems, caused by the hYAK3 imbalance including, but not limited to, neutropenia; cytopenia; anemias, including anemias due to renal insufficiency or to chronic disease, such as autoimmunity or cancer, and drug-induced anemias; polycythemia; and myelosuppression.
Another PSTK of importance in medicine is MK2 protein. Cytokines can induce many of the key features of inflammatory disease and inhibition of their production or mechanism of action would be an appropriate therapeutic approach. Inhibition of p38 MAP kinase has been demonstrated to decrease pro-inflammatory cytokine production including IL-1, TNF-α, IL-6, IL-8 and GMCSF. Inhibiting downstream of p38 may allow for greater selectivity towards these kinases implicated in up-regulation of pro-inflammatory cytokines and may lead to compounds with improved safety profiles. MAPKAP K2 (MK2) lies downstream and is directly activated by p38 MAP kinase. It has been established that MK2 and p38 exist as a complex in the nucleus and that phosphorylation of MK2 by p38 results in the export of this complex from the nucleus to the cytoplasm (Ben-Levy et al., Curr Biol 1998; 8:1049-57). Thus MK2 not only acts as a substrate but also as a determinant of the cellular localization of p38, which is consistent with a role for MK2 in both transcriptional and translational events
Data from the MK2 knock-out mouse has demonstrated an important role for this kinase in pro-inflammatory cytokine production. MK2−/− knock-out mice exhibited a 90% reduction in LPS-induced TNF-α production and were resistant to endotoxic shock. Spleen cells from the MK2−/− mice also demonstrated significant inhibition of the pro-inflammatory cytokines TNF-α, IL-1β, IFN-γ and IL-6 following LPS stimulation (Kotlyarov et al., Nature Cell Biology 1999; 1:94-97). Compounds which are active against MK2 are believed to be useful in the treatment or prevention of rheumatoid arthritis, COPD, asthma, psoriasis, acute neuronal injury, heart failure, stroke, osteoarthritris, and ischemia reperfusion injury.
Compounds of the present invention are found to have activities against hYAK3 and/or MK2 proteins.